Rapid low-cost detection of hepatitis C virus RNA in HCV-infected patients by real-time RT-PCR using SYBR green I

We intend to design and validate a low-cost assay for the detection of hepatitis C virus [HCV] RNA using rapid-cycle RT-PCR. The procedure is performed in a closed system with little risk of contamination allowing PCR and product identification to be performed within one or two hours. A SYBR Green-based real-time RT-PCR for rapid detection of HCV. Amplicon synthesis was monitored continuously by SYBR Green I, which binds to double stranded DMA during PCR. The PCR products were identified by melting curve analysis. Standard sera with known concentrations of HCV RNA and 150 clinical samples were used to validate our assay. The minimum detection level of our assay was less than 50 ID/mL. The results on 100 plasma samples were comparable with commercial assays. This method is useful for rapid qualitative detection of HCV infection and particularly suitable for routine diagnostic applications

Seroprevalence and risk factors of hepatitis a virus infection in Iran: a population based study

In older studies, the seroprevalence of hepatitis A virus infection has been reported to be over 95% in Iranians. Most of these studies were performed on volunteer blood donors. Studies on the general population are sparse. The purpose of this study was to determine the current seroprevalence of hepatitis A virus infection in the general population of Iran. During 2006, 1869 subjects between 18 and 65 years of age were randomly selected from the general population of three Iranian provinces [Tehran, Golestan, and Hormozgan]. Subjects were interviewed and a plasma sample was obtained for serologic testing for anti-hepatitis A virus. Univariate and multivariate analysis was performed to identify risk factors. The seroprevalence of hepatitis A virus in Tehran, Golestan and Hormozgan was 85%, 99%, and 96%, respectively. The overall seroprevalence of hepatitis A virus in the general population of the three provinces studied was 86% and did not differ between the two genders. The prevalence in younger subjects and in urban populations was under 70%. In multivariate analysis, older age, being married, and level of the father's education was associated with hepatitis A virus seropositivity. The seroprevalence of hepatitis A virus still appears to be too elevated for recommending routine vaccination in the general population. However, the trend towards a lower prevalence in younger age groups and people from urban areas points towards the possible benefit of vaccination in these subgroups

use of a sensitive RT-Nested PCR method for detection of Hepatitis C virus

Hepatitis C virus is the major cause of viral hepatitis and its diagnosis in suspected specimens is of great importance. The risk of transfusion- transmitted virus infection is primarily the result of failure in serological screening tests to detect recently infected donors in the pre-seroconversion window period of infection. Therefore, sensitive and accurate diagnosis of HCV prior to antibody production to reduce window period is necessary. In the present study, a sensitive and specific RT-Nested PCR method for detection of a conserved HCV 5 UTR sequence was developed. Two pairs of primers for amplification of the target sequence in two rounds of PCR were selected. The developed RT- Nested PCR assay was performed on HCV-antibody confirmed positive samples as well as negative controls and standard samples. In order to compare the results, One Step RT-PCR kit was used in this study. 25 HCV-positive plasma samples whose positivity were confirmed by ELISA and Western Blot tests, also as well as 10 fold dilutions of a high viral load plasma sample obtained from a HCV-positive patient as standard samples and 25 negative control plasmas from healthy blood donors were collected and tested by this assay. In all of positive samples a 175bp band was observed on agarose gel electrophoresis, but no band could be detected in negative control plasma. Results from developed RT-PCR assay and One Step RT-PCR kit showed a good correlation. According to the results of this study, the developed RT-Nested PCR assay has a good sensitivity and specificity for diagnosis of HCV infection. It has the advantage of viral genome detection prior to seroconversion and can be used to detect HCV infection during window period

comparison between pegylated interferon alpha-2a and standard interferon in combination with ribavirin in treating patients with bleeding disorders and chronic hepatitis C

Patients with bleeding disorders are frequently infected with hepatitis C virus [HCV]. There are few reports on the effect of standard interferon in these patients and no published report on pegylated interferon. The aim of this study was to compare pegylated interferon alpha-2a and standard interferon alpha with ribavirin in patients with bleeding disorders and chronic HCV infection. Consecutive patients referring to a specialized clinic in Tehran were included in the study. The first 37 patients received pegylated interferon [PEGASYS, Hoffmann-La Roche Inc., Basel, Switzerland], 180 microg weekly and the next 38 patients received standard interferon, 3 million units 3 times a week. Both groups also received ribavirin 800 mg daily. Patients were treated for 48 weeks and were followed for 24 weeks. Liver biopsy was not performed due to the potential risks involved in patients with bleeding disorders. 34 patients in each group completed the study. The intention-to-treat sustained viral response was 34% and 62% in the standard interferon and pegylated interferon group, respectively [p=0.02]. Pegylated interferon alpha-2a and ribavirin is almost twice as effective as standard interferon and ribavirin in treating HCV infection in patients with bleeding disorders and is an acceptable treatment option even when histologic data is not available

comparison between Pegylated interferon alpha-2a and standard interferon in combination with ribavirin in treating patients with bleeding disorders and chronic hepatitis C

Patients with bleeding disorders are frequently infected with hepatitis C virus [HCV]. There are few reports on the effect of standard interferon in these patients and no published report on pegylated interferon. The aim of this study was to compare pegylated interferon alpha-2a and standard interferon alpha with ribavirin in patients with bleeding disorders and chronic HCV infection. Consecutive patients referring to a specialized clinic in Tehran were included in the study. The first 37 patients received pegylated interferon [PEGASYS, Hoffmann-La Roche Inc., Basel, Switzerland], 180 microg weekly and the next 38 patients received standard interferon, 3 million units 3 times a week. Both groups also received ribavirin 800 mg daily. Patients were treated for 48 weeks and were followed for 24 weeks. Liver biopsy was not performed due to the potential risks involved in patients with bleeding disorders. 34 patients in each group completed the study. The intention-to-treat sustained viral response was 34% and 62% in the standard interferon and pegylated interferon group, respectively [p=0.02]. Pegylated interferon alpha-2a and ribavirin is almost twice as effective as standard interferon and ribavirin in treating HCV infection in patients with bleeding disorders and is an acceptable treatment option even when histologic data is not available

Pasteurization of IgM-enriched immunoglobulin

Human plasma proteins are important for therapy or prophylaxis of human diseases. Due to the preparation of human plasma proteins from human plasma pools and risk of contamination with human viruses, different viral reduction treatments such as: pasteurization, solvent/detergent, dry heat treatment, steam treatment, beta-propiolactone/UV and nanofiltration have been implemented. As pasteurization can be performed for liquid protein, this method [a 10-hour heat treatment of the aqueous solutions at 60°C] was introduced into the manufacturing procedure of IgM-enriched immunoglobulin, to improve its safety further. The efficiency of this method for inactivation of viruses was evaluated by the use of Foot-and-Mouth Disease Virus [a non-enveloped virus] and Infectious Bovine Rhinotracheitis [IBR] Virus [a lipid-enveloped virus]. Pasteurization inactivated Foot-and-Mouth Disease Virus by 7 log10 and for IBR Virus by 5log10. These findings show a significant added measure of virus safety associated with pasteurization of IgM-enriched immunoglobulin preparation

Preparation of enriched immunoglobulin M and immunoglobin A from human plasma

As IgM and IgA-enriched preparations are needed to complete the immunotherapeutic spectrum, a simple procedure is described for the preparation of IgM and IgA-enriched immunoglobulins. Fraction III which was prepared by cold ethanol fractionation was treated by octanoic acid followed by ethanol precipitation and ion-exchange chromatography using Sephadex DEAE A-50 and 0.1 M tris-0.35M NaC1 buffer, pH 8.1, resulting in recovery of 85% IgM, 84% IgA and 33% IgG. The comparison of our results with immunoglobulins' percentage in plasma indicates that IgM and IgA-enrichment was obtained by three times

Solvent-detergent treatment of IgM-enriched immunoglobulin

Viral safety of human plasma products plays a key role in their safe uses. Solvent- detergent [SD] virus-inactivation method has gained widespread popularity in the manufacture of biological products. This treatment which inactivates lipid-enveloped viruses effectively consists of incubation of a plasma protein solution in the presence of a non-volatile organic solvent and a detergent. In this study, IgM-enriched immunoglobulin was incubated at 24 °C for 6 h under slow stirring in the presence of tri[n-butyl] phosphate [0.3% w/w] as solvent and tween 80 [1% w/w] as detergent. After completion of the inactivation process and removal of the solvent-detergent, the ability of SD-treatment to remove Infectious Bovine Rhinotracheitis [IBR] virus [a lipid-enveloped virus] and Foot-and-Mouth Disease virus [a non-enveloped virus] were evaluated by "virus spiking studies" using a scaled down process. Reduction factor of 4 log was obtained for the SD-treatment of IgM-enriched immunoglobulin spiked with IBR virus. No virus inactivation was observed in the SD-treated IgM-enriched immunoglobulin, spiked with Foot-and-Mouth Disease virus. It was concluded that treatment of IgM-enriched immunoglobulin with TNBP-TWEEN 80 may be considered as an efficient lipid-enveloped virus inactivation step in the manufacture of this product